As I'm working with RIG-I activation, I was looking for an easy way to detectd RIG-I ubiquitination levels in transfected cells. Someone has a method to recommend? I tried with SDS/boiling lysis followed by IP but a smear without any clear band was detected by immunoblotting.
RIPA lysis buffer and 6% Acrylamide Gels for SDS-PAGE are good selection for detection of ubiquited RIG-I and RIG-I by immunoblotting. NEM is necessary in lysis buffer.
Thank you Zhang Im. Have you a detailed protocol for that or conventional IP. Did you co-transfect ubiquitin? Thanks in advantage
Yeah, i have co-transfected RIG-I and Ub. However, i only detect RIG-I conjunct binding with four ubiquitins. This paper has deteced three kinds of RIG-I ubiquitined by TRIM25.
http://www.cell.com/cell-host-microbe/fulltext/S1931-3128(09)00107-3
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